Intervertebral disc degeneration is strongly implicated as a cause of low back pain. Over a 10 year period, more than 130,000 active service members received diagnoses of disc degeneration, with annual incidence rates more than doubling during this time. Current treatment approaches are mostly conservative, and in severe cases, patients may undergo surgical procedures such as spinal fusion, which do not maintain or restore native tissue structure or mechanical function. Mesenchymal stem cells (MSCs) are attractive options for cell-based therapies due to their potential for autologous harvest, expansion, and re-implantation making them highly translational. A key challenge to successful application of MSCs for disc regeneration is the unique biochemical and physical microenvironment cells are exposed to upon implantation. To overcome this challenge, one strategy is preconditioning of MSCs by exposure to hypoxia, soft physical substrates and developmentally-relevant stimuli during monolayer expansion in order to enhance their in vivo performance in applications for disc regeneration. It has also been shown that MSCs isolated from bone marrow are highly heterogeneous, both within and between donors, exhibiting distinct phenotypic characteristics, including sensitivity to microenvironmental stress in culture. Aim 1 will therefore establish the in vitro preconditioning and donor characteristics that maximize the survival and extracellular matrix production of MSCs in the oxygen and nutrient-poor disc microenvironment. Preclinical demonstration of the long term efficacy of disc therapies with respective to structural and functional regeneration, and alleviation of symptoms, using rigorously validated, clinically-relevant outcome measures is critical to their successful future clinical translation. Our team recently established a preclinical goat model of disc degeneration that recapitulates key characteristics of human disc degeneration. Therefore, in Aim 2 we will establish the long term efficacy of a combined hydrogel and optimized stem cell therapy to potentiate structural and functional disc regeneration, and alleviate symptoms, in an established preclinical goat model of disc degeneration. To maximize the clinical impact of our findings we will further refine our goat model to include progressive and clinically relevant functional, imaging, and molecular biomarkers of pain and disability. Finally, in Aim 3 we will take an additional important step towards clinical application of this therapy by establishing that adult human MSCs respond to preconditioning, and undertake transcriptome-wide screening studies to identify unique molecular signatures that predispose MSCs from certain human donors to enhanced performance and response to preconditioning.